Department of Chemistry, University of California, Irvine, Irvine, California 92697, United States.
Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California 92697, United States.
Acc Chem Res. 2020 Oct 20;53(10):2384-2394. doi: 10.1021/acs.accounts.0c00474. Epub 2020 Oct 1.
The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the . The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.
2018 年诺贝尔化学奖表彰了体外进化，包括乔治·史密斯（George Smith）和格雷戈里·温特（Gregory Winter）开发的噬菌体展示技术，该技术可用于对抗体的功能能力进行工程改造，使其成为病毒。这种噬菌体解决了抗体固有的问题，包括成本高、热不稳定性和易于聚集。虽然噬菌体展示技术加速了在各种应用中识别和结合蛋白质的肽和蛋白质基序的发现，但在 21 世纪初，使用完整噬菌体颗粒开发生物传感器在很大程度上仍未得到探索。病毒颗粒的大小为 16.5MDa，由数千种蛋白质组装而成，不能简单地替代任何现有生物传感器架构中的抗体。将病毒纳入生物传感器需要我们回答几个问题：什么样的过程将允许病毒被纳入功能生物亲和层？如何将蛋白质疾病标志物与病毒颗粒的结合电转导产生信号？体液的可变盐浓度会干扰电转导吗？需要一种全新的生物传感器架构和一种将分子与病毒结合的电转导新方案。
本账户描述了 2006 年启动的一个研究计划的重点，该计划回答了这些问题。这些努力最终在 2018 年发明了一种专门设计用于与病毒颗粒接口的生物传感器：VIR 生物电阻传感器（VIR Biosensor）。VIR 是由导电聚合物基质组成的电阻器，其中包含 M13 病毒颗粒。通过在频率上跨越 4 个数量级测量该电阻器的电阻抗，同时测量目标蛋白质的浓度和电阻器浸入的介质的离子电导率。大信号幅度加上 VIR 传感器设计固有的简单性，导致高信噪比（S/N>100）和出色的传感器间重现性。使用这种新设备，我们已经测量了尿液样本中的膀胱癌生物标志物核酸脱糖酶（DJ-1）。这种优化的 VIR 具有非常低的传感器间变异系数，在 DJ-1 结合曲线的范围内为 3-7%，下限为 30pM，涵盖了 4 个数量级的浓度范围。
